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Abstract:

Herein, we describe a direct in vitro method that identifies agents that
are toxic against natural human tissue stem cells. We provide a novel
schedule for culturing any cell population containing homologous tissue
stem cells that allows the number and cell kinetics of tissue stem cells,
transient cells, and terminally differentiated cells within the
population to be monitored. Using the passage schedule together with
determination of a growth curve for the population, one can determine
whether or not an agent is toxic to tissue stem cells, or to transient
cells and/or terminal cells. The same method can also be used to identify
agents that act positively on tissue stem cells and the other specific
cell types.

Claims:

1. An in vitro method of culturing a heterogeneous population of cells
that allows for determination of the effect of an agent on the tissue
stem cells comprising: a. culturing a heterogeneous population of cells
comprising tissue stem cells, transient cells and terminally
differentiated cells; and b. performing sequential passages of the
cultured cells of step a) based on a specific time interval for passage
rather than passage based on cell density, wherein the cells are
sequentially passaged at the specific time interval using the same
dilution factor at each passage such that the cells do not reach more
than 50% confluency at the time for passage, and wherein the cells are
sequentially passaged until at least two passages are performed without
any increase in cell number in the culture prior to next passage; thereby
allowing the number and cell kinetics of tissue stem cells, transient
cells and terminally differentiated cells within the population to be
monitored.

2. The method of claim 1, wherein there is a decline in the cell number
of the culture at the time of passage, as compared to the cell number at
the time of a prior passage, within 6 sequential passages.

3. The method of claim 1, wherein the period of time until at least two
passages are performed without any increase in cell number in the culture
is selected from the group consisting of: less than 100 days; less than
90 days; and less than 80 days.

4. The method of claim 1, wherein the heterogeneous population of cells
cultured in step a) is cultured using a cell number selected from the
group consisting of: less than 50,000 cells/cm2; less than 10,000
cells/cm2; and less than 7,000 cells/cm.sup.2.

5. The method of claim 1, wherein the specific time interval is selected
from the group consisting of: every 108 hours; every 96 hours; every 72
hours; and every 48 hours.

6. The method of claim 1, wherein the dilution factor is selected from
the group consisting of: 1:2; 1:3; 1:5; and 1:10.

7. The method of claim 1, wherein the cell number at the two passages
that are performed without any increase in cell number has declined to a
cell number that is selected from the group consisting of: less than 40%
of the cell number in step a); less than 30% of the cell number in step
a); less than 20% of the cell number in step a); less than 10% of the
cell number in step a); and less than 5% of the cell number in step a).

8. An in vitro method of determining the effect of an agent on a
heterogeneous population of cells comprising tissue stem cells, transient
cells and terminally differentiated cells, comprising: a. culturing a
heterogeneous population of cells comprising tissue stem cells, transient
cells and terminally differentiated cells, b. contacting the cultured
cells of step a) with an agent; c. performing sequential passages of the
cultured cells of step b) based on a specific time interval for passage
rather than passage based on cell density, wherein the cells are
sequentially passaged at the specific time interval using the same
dilution factor at each passage such that the cells do not reach more
than 50% confluency at the time for passage, and wherein the cells are
sequentially passaged until at least two passages are performed without
any increase in cell number in the culture prior to next passage; d.
determine the number of cells in the heterogeneous population at the time
of each passage; e. plotting the number of population doubling versus
time of passage to obtain a growth curve for the heterogeneous
population; and f. comparing the growth curve of step e) to a control
culture that has not been contacted with the agent of step b), wherein a
deviation of the curve of step e) from the control indicates the agent
has either a toxic or a positive effect on tissue stem cells, transient
cells, or terminal cells.

9. The method of claim 8, wherein when the deviation of the curve is due
to a lower amount of population doublings early in the growth curve and
to a faster time to reach the two passages that are performed without any
increase in cell number, the agent is toxic to tissue stem cells.

10. The method of claim 8, wherein when the deviation of the curve is due
to a lower amount of population doublings late in the growth curve, and
the time to reach the two passages that are performed without any
increase in cell number in the culture is similar to the control, the
agent is toxic to transient cells.

11. The method of claim 8, wherein when the deviation of the curve is due
to a higher amount of population doublings in the middle of the growth
curve, and the time to reach the least two passages that are performed
without any increase in cell is similar to the control, the agent has a
positive effect on tissue stem cells.

12. The method of claim 8, wherein the positive effect is an increase in
tissue stem cell number, viability, or function.

13. The method of claim 8, wherein the toxic effect is a decrease in
tissue stem cell number, viability, or function.

14. The method of claim 8, wherein there is a decline in the cell number
of the culture at the time of passage, as compared to the cell number at
the time of a prior passage, with six sequential passages.

15. The method of claim 8, wherein the period of time until at least two
passages are performed without any increase in cell number in the culture
is selected from the group

16. The method of claim 8, wherein the heterogeneous population of cells
cultured in step a) is cultured using a cell number selected from the
group consisting of: less than 50,000 cells/cm2; less than 10,000
cells/cm2; and less than 7,000 cells/cm.sup.2.

17. The method of claim 8, wherein the specific time interval is selected
from the group consisting of: every 108 hours; every 96 hours; every 72
hours; and every 48 hours.

18. The method of claim 8, wherein the dilution factor is selected from
the group consisting of: 1:2; 1:3; 1:5; and 1:10.

19. The method claim 8, wherein the percentage of tissue stem cells in
the population is less than 5%.

20. The method of claim 8, wherein the cell number at the two passages
that are performed without any increase in cell number has declined to a
cell number that is selected from the group consisting of: less than 40%
of the cell number in step a); less than 30% of the cell number in step
a); less than 20% of the cell number in step a); less than 10% of the
cell number in step a); and less than 5% of the cell number in step a).

21. An in vitro method of culturing a heterogeneous population of cells
that allows for determination of the effect of an agent on the tissue
stem cells within the heterogeneous population comprising: a. performing
sequential passaging of a heterogeneous population of cells comprising
tissue stem cells, transient cells, and terminally differentiated cells,
in a manner that the cells in culture cease to divide within a period of
100 days (within 90, 80, 60, days), thereby allowing the number and cell
kinetics of tissue stem cells, transient cells and terminally
differentiated cells to be monitored within the population.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit under 35 U.S.C. 119(e) of U.S.
Provisional Application No. 61/978,013, filed Apr. 10, 2014, which is
herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present application is directed to methods for determining the
effect of an agent on a heterogeneous population of cells comprising
tissue stem cells, transient cells, and terminal cells, e.g. a toxic
effect or a positive effect. The methods involve a unique culturing
scheme where the combination of a cell passage schedule based upon a
strict time interval, and the use of the same dilution in each passage
such that the cell culture experiences a rapid decline in cell number,
enables one to monitor the number and cell kinetics of all three types of
cells, i.e. tissue stem cells, transient cells, and terminal cells. The
cell population using such schedule reaches a point of containing only
terminally differentiated cells (a lack of cell division) faster than the
recommended passaging schedules for primary (freshly isolated) or
cultured cells that contain tissue stem cells, transient cells, and
terminal cells. In particular, the culture has cumulative population
doublings that allow for the ability to monitor the number and cell
kinetics of all three cell types within the heterogeneous population of
cells.

BACKGROUND OF THE INVENTION

[0003] Because tissue stem cells are responsible for renewing and
repairing human tissues, drugs that interfere with their function or
cause their death are particularly toxic. FIG. 1 illustrates the
universal, hierarchal human tissue cell kinetics architecture. Tissue
stem cells (NS) subtend tissue turnover units comprised of many dividing
and differentiating transient amplifying cells (NT) and terminally
differentiated cells (NT-Terminal). As differentiated terminal cells age,
expire, and are loss from a given tissue, they are replaced by the
division of transient cells, which are in turn replaced by the division
of the resident tissue stem cells.

[0004] Despite the importance of tissue stem cells in adverse toxic drug
effects, currently there are no pre-clinical assays for tissue stem cell
toxicity that do not require animals. Even animal testing is indirect, as
it involves evaluating the pathological consequences of tissue stem cell
toxicity (e.g., tissue dysplasia, anemia). Also, animal models are known
to be poor predictors for tissue stem cell toxicity in humans.

[0005] A number of factors conspire to cause the current lack of direct
pre-clinical assays for tissue stem cell toxicity. Because of their
unique place in the universal cell kinetics hierarchy of human tissues,
tissue stem cells are a minute fraction of any human tissue cell
preparation. As a result, they have proven difficult to isolate in
sufficient number or purity to establish reliable assays. For the same
reason, no biomarkers for tissue stem cells are available with sufficient
specificity to quantify tissue stem cells for drug toxicity testing.

[0006] Toxicity against tissue stem cells is one of the most intolerable
forms of drug toxicity, which can lead to drug candidate failure late in
expensive clinical trials, and potentially after marketing. However, the
only currently available pre-clinical tests for detecting human tissue
stem cell toxicity are animal models. Such tests are expensive and often
do not faithfully predict toxic effects in human patients

SUMMARY OF THE INVENTION

[0007] We have identified an in vitro method that uses a distinct schedule
for passaging a population of cells comprising tissue stem cells,
transient cells and terminal cells, by which agents that are toxic
against natural human tissue stem cells present in the population can be
discerned. The method is suitable for culturing any heterogeneous
population of cells comprising homologous tissue stem cells. The cell
kinetics data derived from the schedule allows determination of whether
an agent is toxic to tissue stem cells and/or is toxic to the other
specific cell types that compose the natural tissue cell hierarchy (FIG.
1), i.e. transient cells or terminal cells. The same method can also be
used to identify agents that act positively on tissue stem cells and the
other specific cell types. For example, an increase in growth of or
number of tissue stem cells.

[0008] Accordingly, one aspect of the invention provides methods of
culturing a heterogeneous population of cells that allows for
determination of the effect of an agent on the tissue stem cells. The
method comprises step a) culturing a heterogeneous population of cells
comprising tissue stem cells, transient cells and terminally
differentiated cells; and step b) performing sequential passages of the
cultured cells of step a) based on a specific time interval for passage
rather than, for example, passage based on confluency (cell density). The
cells are sequentially passaged at a given time interval using the same
dilution factor at each passage such that the cells do not reach more
than 50% confluency at the time for passage. This results in a cell
culture that rapidly declines in cell number (e.g. a culture where the
cell number at the time of passage starts declining within 4 passages,
within 5 passages, within 6 passages, within 7 passages, or within 10
passages). An important feature is that the culture declines in cell
number. In one embodiment, the cells are sequentially passaged until at
least two passages are performed without any increase in cell number in
the culture prior to next passage. In an alternative embodiment, the
cells are sequentially passaged until a culture at the time of passage
has less than 40%, less than 30%, less than 20%, or less than 10%, of the
original starting cell number (e.g. prior to containing only terminally
differentiated cells, i.e. prior to two times passage where there is no
increase in cell number at the time of passage). The decline in the cell
number in the heterogeneous culture allows the number and cell kinetics
of all three cell types in the heterogeneous population to be monitored,
(i.e. tissue stem cells, transient cells and terminally differentiated
cells).

[0009] In another aspect, the methods for culturing a heterogeneous
population of cells comprising tissue stem cell, transient cells and
terminal cells, described above, are used to determine the effect of a
test agent on the population of cells. The methods comprise culturing a
heterogeneous population of cells as described in step a} and step b) of
the above paragraph. However, in this aspect, the heterogeneous
population of cells is contacted with a test agent prior to, or during,
the sequential passage as described in steps a) and b) of above
paragraph. To determine the effect of the test agent, the cell number of
the culture is determined at each sequential passage so that the number
of population doubling versus time of passage can be plotted in order to
obtain a growth curve for the heterogeneous population. The growth curve
of the cells contacted with the test agent is then compared to a control
culture that has not been contacted with the agent, wherein a deviation
of the growth curve with the agent from the control growth curve
indicates the agent has either a toxic or a positive effect on tissue
stem cells, transient cells, or terminal cells.

[0010] For example, when the deviation of the curve is due to a lower
amount population doublings early in the growth curve and to a faster
time to reach the two passages that are performed without any increase in
cell number (See e.g. FIG. 7), the agent is toxic to tissue stem cells.
When the deviation of the curve is due to a lower amount population
doublings late in the growth curve, and the time to reach the two
passages that are performed without any increase in cell number in the
culture is similar to the control (See e.g. FIG. 9), the agent is toxic
to transient cells. In addition, when the deviation of the curve is due
to a higher amount of population doublings in the middle of the growth
curve, and the time to reach the least two passages that are performed
without any increase in cell is similar to the control (See e.g. FIG.
12), the agent has a positive effect on tissue stem cells. In one
embodiment of this aspect, the positive effect is an increase in tissue
stem cell number, viability, or function. In another embodiment of this
aspect, the toxic effect is a decrease in tissue stem cell number,
viability, or function.

[0011] In one embodiment of each of the aspects above, there is a decline
in the cell number of the culture at the time of passage within six
sequential passages, as compared to the cell number at the time of a
prior passage. In one embodiment of each of the aspects above, there is a
decline in the cell number of the culture at the time of passage, as
compared to the cell number at the time of a prior passage, within five
sequential passages. In one embodiment of each of the aspects above,
there is a decline in the cell number of the culture at the time of
passage, as compared to the cell number at the time of a prior passage,
within 4 sequential passages.

[0012] In some embodiments of each of the aspects above, the period of
time until at least two passages are performed without any increase in
cell number in the culture is less than 100 days, or less than 90 days,
or less than 80 days.

[0013] In some embodiments of each of the aspects above, the starting cell
culture of step a) has a population cell number that corresponds to
200,000 cells in a 75 cm2 plate or flask, 65,000 cells in a 25
cm2 plate or flask, or 22,000 cells in a 8.3 cm2 plate or
flask. In some embodiments, the starting cell number of the culture is
less than 50,000 cells/cm2, less than 10,000 cells/cm2, or less than
7,000 cells/cm2. In certain embodiments of each of the above
aspects, the cell concentration is greater than 2,600 cells/cm2.

[0014] In some embodiments of each of the aspects above, the specific time
interval is every 108 hours, every 96 hours, every 72 hours, or every 48
hours.

[0015] In some embodiments of each of the aspects above, the dilution
factor is 1:2, is 1:3, is 1:5, or is 1:10.

[0016] In some embodiments of each of the aspects above, the cell number
at the two passages that are performed without any increase in cell
number has declined to less than 40% of the cell number in step a). In
some embodiments of each of the aspects above, the cell number at the two
passages that are performed without any increase in cell number has
declined to less than 30% of the cell number in step a). In some
embodiments of each of the aspects above, wherein the cell number at the
two passages that are performed without any increase in cell number has
declined to less than 20% of the cell number in step a). In some
embodiments of each of the aspects above, the cell number at the two
passages that are performed without any increase in cell number has
declined to less than 10% of the cell number in step a).

[0017] In some embodiments of each of the aspects above, the percentage of
tissue stem cells in the population is less than 5%.

[0018] In some embodiments of each of the aspects above, the heterogeneous
population of cells is obtained from organ tissue.

[0019] In some embodiments of each of the aspects above, the heterogeneous
population of cells is obtained from diseased tissue. For example,
non-limiting examples of diseased tissue include virally infected tissue,
or tissues having a genetic defect.

[0020] In some embodiments of each of the aspects above, the heterogeneous
population of cells are infected, transformed or transfected to produce a
heterogeneous population of cells that are models of a specific disease.
For example the cells may be transformed with a bacterial plasmid,
transfected with viral vectors, or infected with viruses to mimic a
disease state thereby allowing assessment of the effect of a test agent
on tissue stem cells that are associated with disease. In certain
embodiments, nucleic acid is introduced to express proteins related to
the disease. In certain embodiments, nucleic acid is introduced that in
inhibits gene expression, e.g. RNAi or antisense.

[0021] Also provided, is a method of culturing a heterogeneous population
of cells that allows for determination of the effect of an agent on the
tissue stem cells within the heterogeneous population comprising:
performing sequential passaging of a heterogeneous population of cells
comprising tissue stem cells, transient cells, and terminally
differentiated cells, in a manner that the cells in culture cease to
divide within a period of 100 days (within 90, 80, 60, days), thereby
allowing the number and cell kinetics of tissue stem cells, transient
cells and terminally differentiated cells to be monitored within the
population. In one embodiment, the cell number of the culture begins to
decline at the time of passage within six sequential passages, (or within
5 sequential passages, or within 4 sequential passages) as compared to
the cell number present at the time of a prior passage. In one
embodiment, the cells are sequentially passaged until the culture at the
time of passage has less than 40%, less than 30%, less than 20%, or less
than 10%, of the original starting cell number.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The priority application file contains at least one drawing
executed in color. Copies of the priority application with color
drawing(s) will be provided by the Office upon request and payment of the
necessary fee.

[0023] FIG. 1 shows a schematic of the three types of cells present in
tissue and their hierarchal cell kinetics architecture.

[0024] FIG. 2 shows a graph of one embodiment of computer-simulation of
total cell number with culture transfers on the invention schedule and
the conventional schedule employed for human cells. Simulation was
stopped at 85 days. Note how the conventional schedule depends on culture
confluency (i.e., reaching the culture vessel's maximum cell capacity
before each dilution).

[0025] FIG. 3 shows a graph of the corresponding cumulative population
doublings (CPD) output for human liver tissue cells cultured on one
embodiment of the invention schedule compared to culture on the
conventional schedule (based on FIG. 2 analyses).

[0026] FIG. 4 shows a graph of a computer-simulation to illustrate the
differences in the rate of tissue stem cell dilution for the invention
schedule versus the conventional schedule. Note how the conventional
schedule results in stabilization of tissue stem cell fraction, which
precludes the statistical deviation required to distinguish cell
type-specific effects.

[0027] FIG. 5 shows a graph of a computer-simulation of the total cell
number output produced by serial culture of human liver cells. These data
are transformed into cumulative population doubling data for cell
kinetics analyses (culture CPD). Vertical lines denote culture dilution
events.

[0028] FIG. 6 shows a graph of a computer-simulated deconstruction of the
stem cell, transient cell, and terminal cell components of the total cell
data in FIG. 2.

[0029] FIG. 7 shows a graph of a comparison of computer-simulated cell
kinetics data for control serial culture, serial culture with a non-stem
cell toxic agent at its IC50, and serial culture with a stem cell toxic
agent at its IC50.

[0030] FIG. 8 shows a graph of a computer-simulated deconstruction of the
stem cell, transient cell, and terminal cell components of the cell
kinetics data in FIG. 7 for serial culture with a stem cell toxic agent.

[0031] FIG. 9 shows a graph of a comparison of computer-simulated cell
kinetics data for an untreated control serial culture, a serial culture
treated with a transient cell-specific toxic agent at its IC90, and a
serial culture treated with a tissue stem cell-specific toxic agent at
its IC90. CDP, cumulative population doublings.

[0032] FIG. 10 shows a graph of a comparison of computer-simulated cell
kinetics data for an untreated control serial culture, a serial culture
treated with a tissue stem cell-specific toxic agent at its IC90, and a
serial culture treated with an agent toxic for both transient cells and
tissue stem cells at its IC90. CDP, cumulative population doublings.

[0033] FIG. 11 shows a graph of a comparison of computer-simulated cell
kinetics data for an untreated control serial culture, a serial culture
treated with a transient cell-specific toxic agent at its IC90, and a
serial culture treated with a terminal cell-specific toxic agent at its
IC90. CDP, cumulative population doublings.

[0034] FIG. 12 shows a graph of a computer-simulation of the effect of a
tissue stem cell-activating agent on the cumulative population doubling
(CPD) output of the invention passage schedule for a human liver cell
culture enriched for human liver stem cells. In the control condition,
about 70% of human liver tissue stem cell divisions are asymmetric (as
diagrammed in FIG. 1). Approximately 30% of the divisions are symmetric,
producing two stem cells--and as a result no transient cells. The data
shown simulate the effect of an agent that causes 70% of tissue stem cell
divisions to become symmetric, with only 30% remaining asymmetric.

[0039] We have now invented a method of culturing a heterogeneous
population of cells comprising tissue stem cells, transient cells, and
terminal cells that enables the kinetics of all three culture types to be
monitored, e.g. to determine the effect on an agent on each of the three
cell types. In certain embodiments, agents are assessed for a toxic
(negative) effect of an agent on tissue stem cells. In certain
embodiments, agents are assessed for a positive effect of an agent on
tissue stem cells, e.g. agents that increase cell growth, viability, or
cell number of tissue stem cells are desirable.

[0040] The present application is directed to methods for determining the
effect of an agent on a heterogeneous population of cells comprising
tissue stem cells, transient cells, and terminal cells, e.g. a toxic
effect or a positive effect. The methods involve a unique culturing
scheme where the combination of a cell passage schedule based upon a time
interval, and the use of the same dilution in each passage such that the
cell culture experiences a rapid decline in cell number, and reaches the
point of containing only terminally differentiated cells (a lack of cell
division) faster than the recommended passaging schedules for primary
(freshly isolated) or cultured cells containing tissue stem cells,
transient cells, and terminal cells. In particular, the culture has
cumulative population doublings that allow for the ability to monitor the
number and cell kinetics of all three cell types within the heterogeneous
population of cells.

[0041] Embodiments of the invention circumvent the longstanding barriers
of isolation and identification of tissue stem cells. It does so applying
tested drugs to directly treat tissue stem cells in the context of fresh
or previously cultured human tissue cell preparations. The rate-limiting
factor for the long-term cell production of any mammalian cell culture is
directly related to the number, viability, and health of tissue stem
cells in the culture. As shown in FIG. 1, since all transient cells
progress eventually to non-dividing terminal cells, continued cell
production by a mammalian cell culture absolutely depends on the
continued presence of tissue stem cells in the culture.

[0042] Because of the asymmetric self-renewal of tissue stem cells, if a
cell culture is serially passaged, cell production eventually stops
because of the consequential dilution of the tissue stem cell number in
the culture to zero (2-7). Conventional serial passaging involves growing
a cell culture until the culture vessel is replete with cells, e.g.
greater than 70%, greater than 80%, or more confluency. When replete, the
cells are harvested; and a fixed fraction of the harvested cells is
transferred to a new culture vessel. The new culture is allowed to grow
until replete again, and the dilution process is performed again. There
are well known examples of such serial passaging schedules for both human
cells (8) and rodent cells (9). In the case of human tissue cell
cultures, this serial process inevitably leads to a complete stoppage in
new cell production. At the endpoint, the cultures are predicted to
contain only terminal cells. This outcome results from first dilution of
tissue stem cell number to zero, followed by completion of the remaining
transient cells differentiation and production of terminal cells (2).

[0043] We have discovered that by altering the typical serial passage
schedule to create a culture that declines, it is possible to relate the
total cell output of a culture containing tissue stem cells to the
relative number, viability, and quality of tissue stem cells present. In
embodiments of the invention the culturing schedule does not wait for
cell cultures to become replete with cells, (e.g. greater than or equal
to 80% confluency). Instead, the specified schedule of passaging is
maintained no matter what cell number is obtained at the end of each
growth interval. Even when the cell number appears fixed, the dilution is
continued until there are at least two successive passage intervals
without any increase in cell number. The cell kinetics of such a schedule
can be related directly to the number, viability, and health of tissue
stem cells during the duration of the serial passaging. It can also be
related to the cell kinetics activities of transient cells and terminal
cells.

[0044] By comparing the cell kinetics (i.e., total cell number versus time
or cumulative cell population doublings versus time) of control culture
data to drug-treated culture data, it is possible to determine whether an
agent is toxic to, is neutral, or has a positive effect on, tissue stem
cells, transient cells, terminal cells, or any and all combinations of
the three cell types. Control culture data are representative of the same
heterogeneous population of cells not treated with the drug, i.e. the
test agent, e.g. the same heterogeneous population of cells is split into
a control group and a test group for a control culture and a test
culture, which are treated the same except for the presence of dug being
present in the test culture and absence of drug in the control culture.
Alternatively, the control culture data is a computer-simulated curve
determined using the probabilistic stem cell kinetics (PSCK; 6,10) model
(See for example FIGS. 4-12) of asymmetric and symmetric kinetics, the
model being representative of the kinetics of the same population of
cells being tested against the agent.

[0045] The methods of the invention can be used to easily identify agents
that are toxic to tissue stem cells without the need for tissue stem cell
isolation and purification. Conversely, it of course follows that the
same methods can also be used to identify agents that act on tissue stem
cells, transient cells, or terminal cells to increase their division,
viability, or function.

[0046] Embodiments of the invention provide methods of culturing a
heterogeneous population of cells that allows for determination of the
effect of an agent on the tissue stem cells. The method comprises a)
culturing a heterogeneous population of cells comprising tissue stem
cells, transient cells and terminally differentiated cells; and b)
performing sequential passages of the cultured cells of step a) based on
a specific time interval for passage rather than passage based on cell
density. The cells are sequentially passaged at the specific time
interval using the same dilution factor at each passage such that the
cells do not reach more than 50% confluency at the time for passage. This
results in a cell culture that rapidly declines in cell number (e.g. that
starts declining within 4 passages, within 5 passages, or within 10
passages). The cells are sequentially passaged until at least two
passages are performed without any increase in cell number in the culture
prior to next passage. The decline in the cell number in the culture
allows the number and cell kinetics of all three cell types in the
heterogeneous population to be monitored, (i.e. tissue stem cells,
transient cells and terminally differentiated cells).

[0047] In certain embodiments, the period of time until at least two
passages are performed without any increase in cell number in the culture
is less than 100 days, less than 90 days, or less than 80 days. Typical
passage of heterogeneous cell populations involve passaging based on
confluency of the cell culture, because cells prefer to be close to one
another for optimal growth conditions.

[0048] In certain embodiments of the above aspects, the period of time
until at least two passages are performed without any increase in cell
number in the culture is less than 100 days, less than 90 days, or less
than 80 days. Typical passage of heterogeneous cell populations involve
passaging based on confluency of the cell culture as cells prefer to be
close to one another for optimal growth conditions.

[0049] The time interval for passage and the dilution factor can vary
dependent upon the starting cell population in order to obtain a decline
in cell number of the culture at the time of passage, for example, within
4 sequential passages, within 5 sequential passages, within 6 sequential
passages, within sequential 7 passages, or within 10 sequential passages
as compared to the cell number present at the time of a prior passage.
The combination is such that the cell number in the culture at the time
of passage continues to decline after reaching maximum cell number, for
example after reaching maximum cell number at the time of sequential
passage number 3, sequential passage number 4, sequential passage number
5, or sequential passage number 7.

[0050] In one embodiment, the specific time interval used for each
sequential passage of the cell culture (i.e. of a heterogeneous
population of cells comprising tissue stem cells, transient cells and
terminal cells) is every 108 hours. In one embodiment, the specific time
interval used for each sequential passage of the cell culture is every 96
hours. In one embodiment, the specific time interval used for each
sequential passage of the cell culture every 72 hours. In one embodiment,
the specific time interval used for each sequential passage of the cell
culture is every 48 hours.

[0051] In certain embodiments the dilution factor at the given time
interval is 1:2, or 1:3, or 1:5, or 1:10. It should be noted that any
combination of time interval and dilution factor can be used as long as
during the time of passage the culture does not reach more than 50%
confluency at any passage. This method ensures that the culture will
eventually present with a declining cell number and containing only
terminally differentiated cells (lack of cell division) within a period
of time less than 100 days, for example within 100 days, 90 days, 80
days, or 70 days. The recommended passage schedule based on confluency
for a heterogeneous population of cells comprising tissue stem cell,
transient cells and terminal cells typically results in a culture that
contains only terminally differentiated cells (lack of cell division)
greater than 100 days, e.g. 150 days. In addition, the recommended
passage schedule based on confluency for a heterogeneous population of
cells comprising tissue stem cell, transient cells and terminal cells
results in a population of cells throughout the 80, 90 or 100 days, does
not allow for discerning the number and cell kinetics of the three cell
types, tissue stem cells, transient cells, and terminal cells.

[0052] We have determined that, if one monitors the population growth
kinetics in a declining culture, the number and cell kinetics of all
three cell types can be discerned. In certain embodiments, the cell
number at the two passages that are performed without any increase in
cell number has declined to less than 40% of the starting cell number of
the heterogeneous cell culture. In one embodiment, the cell number at the
two passages that are performed without any increase in cell number has
declined to less than 30% of the starting cell number of the
heterogeneous cell culture. In one embodiment the cell number at the two
passages that are performed without any increase in cell number has
declined to less than 20% of the starting cell number of the
heterogeneous cell culture. In one embodiment, the cell number at the two
passages that are performed without any increase in cell number has
declined to less than 10% of the starting cell number of the
heterogeneous cell culture.

[0053] As mentioned above, the starting number of cells can vary. However,
it should be that the cells do not reach more than 50% confluency at any
time interval of passage to ensure a rapid decline in cell culture and
the ability to obtain a kinetic growth curve during the decline phase.
Examples of appropriate cell numbers include, for example, 200,000 cells
in a 75 cm2 plate or flask, 65,000 cells in a 25 cm2 plate or
flask, or 22,000 cells in a 8.3 cm2 plate or flask. In some
embodiments, the starting cell number of the culture is less than 50,000
cells/cm2, less than 10,000 cells/cm2, or less than 7,000
cells/cm2. In certain embodiments, the cell concentration is greater
than 2,600 cells/cm2.

[0054] In one embodiment, a method of culturing a heterogeneous population
of cells that allows for determination of the effect of an agent on the
tissue stem cells within the heterogeneous population is provided that
comprises performing sequential passaging of a heterogeneous population
of cells comprising tissue stem cells, transient cells, and terminally
differentiated cells, in a manner that the cells in culture cease to
divide within a period of 100 days (or within 90, within 80,or within 60
days), thereby allowing the number and cell kinetics of tissue stem
cells, transient cells and terminally differentiated cells to be
monitored within the population. The culture ceases to divide earlier
than observed with traditional passaging schedules because the culture is
in a decline phase throughout a majority of the passages. It is the
decline stages that allow all three kinetic cell types to be monitored
for number and cell kinetics. In one embodiment, the cell number of the
culture begins to decline at the time of passage within six sequential
passages, (or within 5 sequential passages, or within 4 sequential
passages) as compared to the cell number present at the time of a prior
passage. In one embodiment, the cells are sequentially passaged until the
culture at the time of passage has reached less than 40%, less than 30%,
less than 20%, or less than 10%, of the original starting cell number.

[0055] In methods of the invention, the starting population is a
heterogeneous population of cells. As used herein a "heterogeneous
population of cells" is a cell population that contains three kinetic
cell types, tissue stem cells, transient cells and terminal cells. These
cells exhibit the kinetic hierarchy as depicted in FIG. 1. In certain
embodiments, the percentage of tissue stem cells in the population is
less than 5%, less than 10%, less than 20%, or less than 30%.

[0056] As used herein, the term "tissue stem cell" refers to somatic stem
cells that are derived from adult tissues and herein are sometimes
referred to as simply as stem cells, or adult stem cells. Somatic stem
cells include cells in tissues that divide to produce the differentiated
cells of the tissue, while maintaining their own undifferentiated stem
cell properties. The term "tissue stem cell" is used to refer to any
multipotent or unipotent stem cell derived from non-embryonic tissue,
including fetal, juvenile, and adult tissue. Tissue stem cells have been
isolated from a wide variety of adult tissues including blood, bone
marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and
cardiac muscle. Each of these tissue stem cells can be characterized
based on gene expression, factor responsiveness, tissue reconstitution,
chromosome segregation, and cell culture kinetics. Exemplary tissue stem
cells include liver stem cells, hair follicle stem cells, neural stem
cells, neural crest stem cells, mesenchymal stem cells, hematopoietic
stem cells, and pancreatic stem cells. As indicated above, tissue stem
cells have been found resident in virtually every tissue. "Adult stem
cell," "somatic stem cell," and "tissue stem cell" are used
interchangeably.

[0057] The term "multipotent" when used in reference to a "multipotent
cell" refers to a cell that is able to differentiate into some but not
all of the cells derived from all three germ layers; or into multiple
cell types that constitute a single type of tissue or organ. Thus, a
multipotent cell is a partially differentiated cell with respect to the
earliest embryonic cells. Multipotent cells are well known in the art,
and examples of multipotent cells include tissue stem cells, such as for
example, hematopoietic stem cells, neural stem cells, hair follicle stem
cells, liver stem cells, etc. Multipotent means a stem cell can form many
types of cells in a given lineage, but not cells of other lineages. For
example, a multipotent blood stem cell can form the many different types
of blood cells (red, white, platelets, etc.), but it cannot form neurons;
cardiovascular progenitor cell differentiate into specific mature
cardiac, pacemaker, smooth muscle, and endothelial cell types;
pancreas-derived multipotent progenitor colonies produce cell types of
pancreatic lineage (cells that produces insulin, glucagon, amylase or
somatostatin) and neural lineage (cells that are morphologically
neuron-like, astrocytes-like or oligodendrocyte-like). As described
above, the tissue stem cell can be multipotent or unipotent (produces one
differentiated cell type like limbal stem cells that make corneal
epithelium cells).

[0059] During asymmetric cell kinetics, one daughter cell divides with the
same kinetics as its stem cell parent, while the second daughter gives
rise to a differentiating non-dividing cell lineage. The second daughter
may differentiate immediately; or depending on the tissue, it may undergo
a finite number of successive symmetric divisions to give rise to a
larger pool of differentiating cells. The second daughter and its
dividing progeny are called transit cells (Loeffler and Potten, Stem
cells and cellular pedigrees--a conceptual introduction. In Stem Cells,
C. S. Potten, ed., San Diego, Calif.: Harcourt Brace & Co., pp. 1-28
(1997)), herein referred to as "transient cells". Transit cell divisions
ultimately result in mature, differentiated, terminally arrested cells,
herein referred to as "terminally differentiated cells", also known as
"terminal cells." In tissues with high rates of cell turnover, the
endpoint for differentiated terminal cells is programmed cell death by
apoptosis.

[0060] Asymmetric cell kinetics evolved in vertebrates as a mechanism to
insure tissue cell renewal while maintaining a limited set of stem cells
and constant adult body mass. Mutations that disrupt asymmetric cell
kinetics are an absolute requirement for the formation of a clinically
significant tumor mass (Cairns, Mutation selection and the natural
history of cancer. Nature 255, 197-200. (1975)). In many ways, asymmetric
cell kinetics provide a critical protective mechanism against the
emergence of neoplastic growths that are life threatening.

[0061] In culture, continued asymmetric cell kinetics of explanted cells
are a major obstacle to their expansion in vitro. Ongoing asymmetric cell
kinetics results in dilution and loss of an initial relatively fixed
number of stem cells by the accumulation of much greater numbers of their
terminally differentiating progeny. If a sample includes both
exponentially growing cells as well as somatic stem cells, the
multiplication of the exponentially growing cells will rapidly overwhelm
the somatic stem cells, leading to their dilution.

[0064] The heterogeneous cell population of the present invention may be
isolated from tissue of an adult mammal, preferably a human. Cells can be
obtained from donor tissue, such as donor skin or other organs, by
dissociation of individual cells from the connecting extracellular matrix
of the tissue. Tissue is removed using a sterile procedure, and the cells
are dissociated using any method known in the art including treatment
with enzymes such as trypsin, collagenase, and the like, or by using
physical methods of dissociation such as with a blunt instrument. The
heterogeneous cell population may also be obtained from bodily fluids;
including, but not limited to, blood, umbilical cord, spinal fluid,
pleural fluid, and lymphatic fluid.

[0066] Also useful in methods of the invention are cell culture systems
that contain a heterogeneous population of cells including stem cells,
transient cells, and terminally differentiated cells. Such systems are
known to those of skill in the art and include, but are not limited to
those described in, U.S. patents and publications 20140193910; U.S. Pat.
Nos. 8,759,098; 8,404,481; 7,883,891; 7,867,712; 7,824,912; 7,655,465;
7,645,610, and 20030133918; which are herein incorporated by reference in
their entirety. Thus, known in the art are cell culture system for all
types of tissues including, but not limited to, human liver, skin, heart,
kidney, lung, hematopoietic, hair follicle, muscle, and pancreatic cells.

[0067] In certain embodiments, the heterogeneous population of cells is
obtained from diseased tissue. For example, non-limiting examples of
diseased tissue include virally infected tissue, or tissues having a
genetic defect that results in disease, or e.g. cancerous tissue, or
e.g., many common diseases and disorders like heart failure, diabetes,
autism, stroke, or kidney disease. Diseased tissue can be isolated to
obtain the heterogeneous population of cells by isolating tissue from a
subject having the disease or disorder of interest. In certain
embodiments, the heterogeneous population of cells is obtained from a
patient having cancer, e.g. bone marrow cancer, leukemia, etc., or cells
isolated from a tumor, or growth.

[0068] In an alternative embodiment, a heterogeneous population of cells
is infected, transformed, or transfected to produce a heterogeneous
population of cells that is a model of a specific disease. For example
the cells may be transformed with a bacterial plasmid, transfected with
viral vectors, or infected with viruses, in order to mimic a disease
state, thereby allowing assessment of the effect of a test agent on
tissue stem cells that are associated with disease. Cells infected with
any virus can be studied, including but not limited to the following
viruses: Retroviruses, Human immunodeficiency virus (HIV), or
Cytomegalovirus, Adenoviruses, Herpesviruses, Poxviruses, Parvoviruses,
Reoviruses, Picornaviruses, Togaviruses, Orthomyxoviruses, and
Rhabdoviruses etc.

[0072] As used herein, the term "passage" refers to diluting the cell
culture, whether it be on plates, or in suspension, and re-culturing
(re-plating) the diluted cells. For example, for passage of cells, the
cells are removed from their tissue culture dish (e.g. by treatment with
trypsin) or flask and diluted so that they can be re-plated (on plates or
in a flask) and allowed to continue to grow.

[0073] Any medium can be used that is capable of supporting cell growth,
including HEM, DMEM, RPMI, F-12, and the like, containing supplements
which are required for cellular metabolism such as glutamine and other
amino acids, vitamins, minerals and useful proteins such as transferrin
and the like. Medium may also contain antibiotics to prevent
contamination with yeast, bacteria and fungi such as penicillin,
streptomycin, gentamicin and the like. In some cases, the medium may
contain serum derived from bovine, equine, chicken and the like. Serum
can contain xanthine, hypoxanthine, or other compounds that enhance
guanine nucleotide biosynthesis, although generally at levels below the
effective concentration to suppress asymmetric cell kinetics. In one
embodiment, for the cell culture the medium and serum contain levels
below the effective concentration to suppress asymmetric cell kinetics.
In one embodiment, the culture medium is a defined culture medium
comprising a mixture of DMEM, F12, and a defined hormone and salt
mixture.

[0074] The culture medium can be supplemented with a
proliferation-inducing growth factor(s). As used herein, the term "growth
factor" refers to a protein, peptide or other biological molecule having
a growth, proliferative, differentiating, or trophic effect on stem cells
or other cell types. Growth factors that may be used include any trophic
factor that promotes cells to proliferate, including any molecule that
binds to a receptor on the surface of the cell to exert a trophic, or
growth-inducing effect on the cell. Examples of proliferation-inducing
growth factors include, but are not limited to, EGF, amphiregulin, acidic
fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor
(bFGF or FGF-2), transforming growth factor alpha (TGFα), and
combinations thereof Growth factors are usually added to the culture
medium at concentrations ranging between about 1 fg/ml to 1 mg/ml.
Concentrations between about 1 to 100 ng/ml are usually sufficient.
Simple titration experiments can be easily performed to determine the
optimal concentration of a particular growth factor. In one preferred
embodiment, epidermal growth factor is used.

[0076] Conditions for culturing should be close to physiological
conditions. The pH of the culture medium should be close to physiological
pH, preferably between pH 6-8, more preferably between about pH 7 to 7.8,
with pH 7.4 being most preferred. Physiological temperatures range
between about 30° C. to 40° C. Cells are preferably
cultured at temperatures between about 32° C. to about 38°
C., and more preferably between about 35° C. to about 37°
C.

[0077] Methods are provided that allow for determination of the effect of
an agent on a heterogeneous population of cells comprising tissue stem
cells, transient cells and terminally differentiated cells. The methods
comprises culturing the heterogeneous population of cells comprising
tissue stem cells, transient cells and terminally differentiated cells,
as described above, herein, and contacting the cultured cells with an
agent. Sequential passages of the cultured cells are then preformed based
on a specific time interval for passage rather than passage based on cell
density, wherein the cells are sequentially passaged at the specific time
interval using the same dilution factor at each passage such that the
cells do not reach more than 50% confluency at the time for passage until
at least two passages are performed without any increase in cell number
in the culture prior to next passage.

[0078] At the time of each passage the number of cells in the
heterogeneous population is determined so that the number of population
doubling versus time of passage can be plotted to obtain a growth curve
for the heterogeneous population. The growth curve is then compared to
the growth curve of a control isogenic culture that has not been
contacted with the test agent. A deviation of the growth curve in the
presence of the test agent from the control growth curve indicates the
agent has either a toxic or a positive effect on tissue stem cells,
transient cells, or terminal cells.

[0079] An agent can positively affect tissue stem cells or transient cells
if there is an increase in tissue stem/transient cell number, viability,
or function. While an agent that affects a decrease in cell number,
viability, or function, of tissue stem cells, transient cell, or terminal
cells, is said to have a toxic effect.

[0080] In an alternative embodiment, rather than passaging the cells until
at least two passages result in no increase in cell number, the cells are
passaged until the culture has less than 40%, 30%, 20%, or 10% of the
cell number initially plated within 100 days, within 90 days or within 80
days of sequential passage.

[0081] As used herein, the term "contacting" or "contact" in connection
with contacting the heterogeneous cell population (e.g. freshly isolated
cells or cultured cells) with a test agent, includes subjecting the cells
to a culture media, which comprises the compound (test agent). In certain
embodiments, the contacting can occur prior to isolation of the cells
from the subject, or tissue, e.g. by administration of an agent to a
subject. Though a benefit of the present invention is that it can serve
as a purely in vitro method. In some embodiments, the contacting with the
agent is only done prior to the sequential passaging. In one embodiment,
the agent is provided to media throughout the growth curve analysis, e.g.
at every sequential passage. In some embodiments, at least two passages,
or at least three, or at least four passages and so on. Those of skill in
the art can vary the time period for contacting the cells and/or the
concentration of the agent in order to obtain a suitable growth curve for
analysis.

[0082] Any test agent can be used in methods of the invention. As used
herein, the terms "test compound" or "test agent" are used
interchangeably and refers to compounds and/or compositions that are to
be tested for their ability to stimulate growth or viability of tissue
stem cells, transient cells etc., or to identify agents that are toxic to
such cells. The test agents can include a wide variety of different
compounds, including known drugs and unknown drugs, including chemical
compounds and mixtures of chemical compounds, e.g., small molecules, e.g.
small organic or inorganic molecules; saccharines; oligosaccharides;
polysaccharides; biological macromolecules, e.g., peptides, proteins, and
peptide analogs and derivatives; peptidomimetics; nucleic acids;
antibodies, nucleic acid analogs and derivatives; an extract made from
biological materials such as bacteria, plants, fungi, or animal cells;
animal tissues; naturally occurring or synthetic compositions; and any
combinations thereof

[0083] As used herein, the term "small molecule" refers to in organic or
organic compounds. However, small molecules typically are characterized
in that they contain several carbon-carbon bonds, and have a molecular
weight of less than 5000 Daltons (5 kD), preferably less than 3 kD, still
more preferably less than 2 kD, and most preferably less than 1 kD.

[0084] The number of possible test agents runs into millions. Of
particular interest are compounds that have been deemed useful for the
treatment of a disease in in vitro studies or in in vivo studies, e.g.
therapeutic compounds. In certain embodiments, the compounds can be
screened in vitro before animal model testing is even attempted thereby
reducing failures at the stage of animal testing for the therapeutic
agent. In addition, using the methods described herein, a compound
library can be compiled of all compounds that show either an increase in
growth and viability of tissue stem cells (and/or transient cells), or
that are neutral to tissue stem cells and transient cells, that can be
used as test agents in therapeutic screens.

[0085] All therapeutic compounds are of interest, and include, but are not
limited to, agents for treatment of cancer, viral infections, bacterial
infections, fungus infections, heart disease, liver disease,
cardiovascular disease, obesity, for facilitation of wound healing, for
treatment of pain, allergies, inflammation, for treatment of genetic
disorders (e.g. hemophilia, cystic fibrosis, neurodegeneration), for
treatment of diabetes, high blood pressure, and the like.

[0087] Test agents can be small molecule compounds, e.g. methods for
developing small molecule, polymeric and genome based libraries are
described, for example, in Ding, et al. J Am. Chem. Soc. 124: 1594-1596
(2002) and Lynn, et al., J. Am. Chem. Soc. 123: 8155-8156 (2001).
Commercially available compound libraries can be obtained from, e.g.,
ArQule, Pharmacopia, graffinity, Panvera, Vitas-M Lab, Biomol
International and Oxford. These libraries can be screened using the
screening devices and methods described herein. Chemical compound
libraries such as those from NIH Roadmap, Molecular Libraries Screening
Centers Network (MLSCN) can also be used.

[0088] Generally, agents can be tested at any concentration that can
modulate growth relative to a control over an appropriate time period.
Typically a range of concentrations is tested for any given agent. In
some embodiments, compounds are tested at concentration in the range of
about 0.1 nM to about 1000 mM. In one embodiment, the compound is tested
in the range of about 0.1 μM to about 20 μM, about 0.1 μM to
about 10 μM, or about 0.1 μM to about 5 μM. In one embodiment,
compounds are tested at 1 μM.

[0089] In certain embodiments, a toxicity profile is obtained by testing a
range of concentrations on isogenic populations of cells and plotting the
data to determine the LD50 (the dose lethal to 50% of the population)
and/or the ED50 (the dose positively effective in 50% of the population).
The dose ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio LD50/ED50. Compositions that
exhibit large therapeutic indices are preferred.

[0090] When determining the effect by the methods described herein, it may
be desirable to use a positive control, for example a drug whose effect
on tissue stem cell kinetics (FIG. 1) is already known. Table 1 provides
just an example of such drugs, there are many drugs known to those of
skill in the art that have an effect on tissue stem cell kinetics.

[0091] Depending upon the particular embodiment being practiced, the test
agents can be provided free in solution, or may be attached to a carrier,
or a solid support.

[0092] In certain embodiments, the test agents are combined, formulated
with a "pharmaceutically acceptable carrier." As used here, the term
"pharmaceutically acceptable" refers to those compounds, materials,
compositions, and/or dosage forms which are, within the scope of sound
medical judgment, suitable for use in contact with the tissues of human
beings and animals without excessive toxicity, irritation, allergic
response, or other problem or complication, commensurate with a
reasonable benefit/risk ratio.

[0093] As used here, the term "pharmaceutically-acceptable carrier" means
a pharmaceutically-acceptable material, composition or vehicle, such as a
liquid or solid filler, diluent, excipient, manufacturing aid (e.g.,
lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or
solvent encapsulating material, involved in carrying or transporting the
subject compound from one organ, or portion of the body, to another
organ, or portion of the body. Each carrier must be "acceptable" in the
sense of being compatible with the other ingredients of the formulation
and not injurious to the patient. Some examples of materials which can
serve as pharmaceutically-acceptable carriers include: (1) sugars, such
as lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, methylcellulose, ethyl cellulose,
microcrystalline cellulose and cellulose acetate; (4) powdered
tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as
magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such
as cocoa butter and suppository waxes; (9) oils, such as peanut oil,
cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such
as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)
esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering
agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic
acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's
solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)
polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,
such as polypeptides and amino acids (23) serum component, such as serum
albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)
other non-toxic compatible substances employed in pharmaceutical
formulations. Wetting agents, coloring agents, release agents, coating
agents, sweetening agents, flavoring agents, perfuming agents,
preservative and antioxidants can also be present in the formulation.

[0094] The amount of agent that is combined with a carrier material to
produce a single dosage form will generally be that amount of the
compound that produces a therapeutic effect. Generally out of one hundred
percent, this amount will range from about 0.1% to 99% of compound,
preferably from about 5% to about 70%, most preferably from 10% to about
30% of the composition.

[0095] When one observes a deviation in the kinetic growth curve in the
presence of the test agent then an effect of the agent is indicated,
either a positive or a toxic effect. By analyzing where the deviation
occurs within the curve one can discern if the effect of the agent is on
tissue stem cells, transient cells, or terminally differentiated cells.
Example deviations between the growth curves in the presence and absence
of a test agent that can be observed are described in the Example 2. For
example, when the deviation of the curve is due to a lower amount
population doublings early in the growth curve and to a faster time to
reach the two passages that are performed without any increase in cell
number (See e.g. FIG. 7), the agent is toxic to tissue stem cells.
However, when the deviation of the curve is due to a lower amount
population doublings late in the growth curve, and the time to reach the
two passages that are performed without any increase in cell number in
the culture prior to next passage (See e.g. FIG. 9) is similar to the
control, the agent is toxic to transient cells. In addition, when the
deviation of the curve is due to a higher amount of population doublings
in the middle of the growth curve, and the time to reach the two passages
are performed without any increase in cell number (is similar to the
control, the agent has a positive effect on tissue stem cells (See e.g.
FIG. 12). Deviations in the curves can also differentiate between
toxicity to terminal or transient cells (See FIG. 11), or when an agent
is toxic to both transient and stem cells (See FIG. 10).

[0096] To determine what is early in the growth phase, late in the growth
phase, and what is in the middle of the growth phase, the skilled artisan
divides the total days in the growth curve into three equal parts, a
first "early phase", a second "middle phase", and a third "late phase."
For example, an 87 day curve is split into three phases of 29 days;
within day 1-29 early phase, within day 30-58 middle phase, and within
day 59-87 late phase.

[0097] Unless otherwise explained, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this disclosure belongs.

[0098] The term "statistically significant" or "significantly" refers to
statistical significance and generally means a two standard deviation
(2SD) below normal, or lower, concentration of the marker. The term
refers to statistical evidence that there is a difference. It is defined
as the probability of making a decision to reject the null hypothesis
when the null hypothesis is actually true. The decision is often made
using the p-value.

[0099] The terms "enriching" or "enriched" are used interchangeably herein
and mean that the yield (fraction) of cells of one type is increased by
at least 10% over the fraction of cells of that type in the starting
culture or preparation.

[0100] In certain embodiments, analysis of the data obtained from the
methods described herein is implemented by computer systems. Accordingly,
also provided are computer readable mediums and computer systems
comprising data modules, storage modules, and comparison modules, that
enable plotting of the growth curves, and enable the comparison of growth
curves treated and not treated with agent. The various derivations of the
curve can then signal through a signaling module, if and how the test
agent is toxic to cells.

[0101] In one embodiment, when the deviation of the curve is due to a
lower amount of population doublings early in the growth curve and to a
faster time to reach the two passages that are performed without any
increase in cell number, the signaling module will signal the agent is
toxic to tissue stem cells.

[0102] In one embodiment, when the deviation of the curve is due to a
lower amount of population doublings late in the growth curve, and the
time to reach the two passages that are performed without any increase in
cell number in the culture is similar to the control, the signaling
module will signal the agent is toxic to transient cells.

[0103] In one embodiment, when the deviation of the curve is due to a
higher amount of population doublings in the middle of the growth curve,
and the time to reach the least two passages that are performed without
any increase in cell is similar to the control, the signaling module will
signal the agent has a positive effect on tissue stem cells.

[0104] In one embodiment, the signaling module will signal a positive
effect that is due to an increase in tissue stem cell number, viability,
or function.

[0105] In one embodiment, the signaling module will signal a toxic effect
that is due to a decrease in tissue stem cell number, viability, or
function.

[0106] As used herein the term "comprising" or "comprises" is used in
reference to compositions, methods, and respective component(s) thereof,
that are essential to the invention, yet open to the inclusion of
unspecified elements, whether essential or not.

[0107] As used herein the term "consisting essentially of" refers to those
elements required for a given embodiment. The term permits the presence
of additional elements that do not materially affect the basic and novel
or functional characteristic(s) of that embodiment of the invention.

[0108] The term "consisting of" refers to compositions, methods, and
respective components thereof as described herein, which are exclusive of
any element not recited in that description of the embodiment.

[0110] Other than in the operating examples, or where otherwise indicated,
all numbers expressing quantities of ingredients or media conditions used
herein should be understood as modified in all instances by the term
"about." The term "about" when used in connection with percentages may
mean±1%.

[0111] The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word "or" is
intended to include "and" unless the context clearly indicates otherwise.
Although methods and materials similar or equivalent to those described
herein can be used in the practice or testing of this disclosure,
suitable methods and materials are described below. The abbreviation,
"e.g." is derived from the Latin exempli gratia, and is used herein to
indicate a non-limiting example. Thus, the abbreviation "e.g." is
synonymous with the term "for example."

[0112] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such publications
that might be used in connection with the present invention. These
publications are provided solely for their disclosure prior to the filing
date of the present application. Nothing in this regard should be
construed as an admission that the inventors are not entitled to antedate
such disclosure by virtue of prior invention or for any other reason. All
statements as to the date or representation as to the contents of these
documents is based on the information available to the applicants and
does not constitute any admission as to the correctness of the dates or
contents of these documents.

[0113] It should be understood that this invention is not limited to the
particular methodology, protocols, and reagents, etc., described herein
and as such may vary. The terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to limit the
scope of the present invention, which is defined solely by the claims.

[0114] In some embodiments of the present invention may be defined in any
of the following numbered paragraphs:

[0115] Paragraph 1. An in vitro method of culturing a heterogeneous
population of cells that allows for determination of the effect of an
agent on the tissue stem cells comprising: a) culturing a heterogeneous
population of cells comprising tissue stem cells, transient cells and
terminally differentiated cells; and b) performing sequential passages of
the cultured cells of step a) based on a specific time interval for
passage rather than passage based on cell density, wherein the cells are
sequentially passaged at the specific time interval using the same
dilution factor at each passage such that the cells do not reach more
than 50% confluency at the time for passage, and wherein the cells are
sequentially passaged until at least two passages are performed without
any increase in cell number in the culture prior to next passage; thereby
allowing the number and cell kinetics of tissue stem cells, transient
cells and terminally differentiated cells within the population to be
monitored.

[0116] Paragraph 2. The method of paragraph 1, wherein there is a decline
in the cell number of the culture at the time of passage, as compared to
the cell number at the time of a prior passage, within 6 sequential
passages.

[0117] Paragraph 3. The method of paragraph 1, wherein the period of time
until at least two passages are performed without any increase in cell
number in the culture is less than 100 days.

[0118] Paragraph 4. The method of paragraph 1, wherein the period of time
until at least two passages are performed without any increase in cell
number in the culture is less than 90 days.

[0119] Paragraph 5. The method of paragraph 1, wherein the period of time
until at least two passages are performed without any increase in cell
number in the culture is less than 80 days.

[0120] Paragraph 6. The method of paragraph 1, wherein less than 50,000
cells/cm2 are cultured in step a).

[0121] Paragraph 7. The method of paragraph 1, wherein less than 10,000
cells/cm2 are cultured in step a).

[0122] Paragraph 8. The method of paragraph 1, wherein less than 7,000
cells/cm2 are cultured in step a).

[0123] Paragraph 9. The method of paragraph 1, wherein the specific time
interval is every 108 hours.

[0124] Paragraph 10. The method of paragraph 1, wherein the specific time
interval is every 96 hours.

[0125] Paragraph 11. The method of paragraph 1, wherein the specific time
interval is every 72 hours.

[0126] Paragraph 12. The method of paragraph 1, wherein the specific time
interval is every 48 hours.

[0131] Paragraph 17. The method of paragraph 1, wherein the cell number at
the two passages that are performed without any increase in cell number
has declined to less than 40% of the cell number in step a).

[0132] Paragraph 18. The method of paragraph 1, wherein the cell number at
the two passages that are performed without any increase in cell number
has declined to less than 30% of the cell number in step a).

[0133] Paragraph 19. The method of paragraph 1, wherein the cell number at
the two passages that are performed without any increase in cell number
has declined to less than 20% of the cell number in step a).

[0134] Paragraph 20. The method of paragraph 1, wherein the cell number at
the two passages that are performed without any increase in cell number
has declined to less than 10% of the cell number in step a).

[0135] Paragraph 21. The method paragraph 1, wherein the percentage of
tissue stem cells in the population is less than 5%.

[0136] Paragraph 22. An in vitro method of determining the effect of an
agent on a heterogeneous population of cells comprising tissue stem
cells, transient cells and terminally differentiated cells, comprising:
a) culturing a heterogeneous population of cells comprising tissue stem
cells, transient cells and terminally differentiated cells, b) contacting
the cultured cells of step a) with an agent; c) performing sequential
passages of the cultured cells of step b) based on a specific time
interval for passage rather than passage based on cell density, wherein
the cells are sequentially passaged at the specific time interval using
the same dilution factor at each passage such that the cells do not reach
more than 50% confluency at the time for passage, and wherein the cells
are sequentially passaged until at least two passages are performed
without any increase in cell number in the culture prior to next passage;
d) determine the number of cells in the heterogeneous population at the
time of each passage; e) plotting the number of population doubling
versus time of passage to obtain a growth curve for the heterogeneous
population; and f) comparing the growth curve of step e) to a control
culture that has not been contacted with the agent of step b), wherein a
deviation of the curve of step e) from the control indicates the agent
has either a toxic or a positive effect on tissue stem cells, transient
cells, or terminal cells.

[0137] Paragraph 23. The method of paragraph 22, wherein when the
deviation of the curve is due to a lower amount of population doublings
early in the growth curve and to a faster time to reach the two passages
that are performed without any increase in cell number, the agent is
toxic to tissue stem cells.

[0138] Paragraph 24. The method of paragraph 22, wherein when the
deviation of the curve is due to a lower amount of population doublings
late in the growth curve, and the time to reach the two passages that are
performed without any increase in cell number in the culture is similar
to the control, the agent is toxic to transient cells.

[0139] Paragraph 25. The method of paragraph 22, wherein when the
deviation of the curve is due to a higher amount of population doublings
in the middle of the growth curve, and the time to reach the least two
passages that are performed without any increase in cell is similar to
the control, the agent has a positive effect on tissue stem cells.

[0140] Paragraph 26. The method of paragraph 22, wherein the positive
effect is an increase in tissue stem cell number, viability, or function.

[0141] Paragraph 27. The method of paragraph 22, wherein the toxic effect
is a decrease in tissue stem cell number, viability, or function.

[0142] Paragraph 28. The method of paragraph 22, wherein there is a
decline in the cell number of the culture at the time of passage, as
compared to the cell number at the time of a prior passage, with six
sequential passages.

[0143] Paragraph 29. The method of paragraph 22, wherein the period of
time until at least two passages are performed without any increase in
cell number in the culture is less than 100 days.

[0144] Paragraph 30. The method of paragraph 22, wherein the period of
time until at least two passages are performed without any increase in
cell number in the culture is less than 90 days.

[0145] Paragraph 31. The method of paragraph 22, wherein the period of
time until at least two passages are performed without any increase in
cell number in the culture is less than 80 days.

[0146] Paragraph 32. The method of paragraph 22, wherein less than 50,000
cells/cm2 are cultured in step a).

[0147] Paragraph 33. The method of paragraph 22, wherein less than 10,000
cells/cm2 are cultured in step a).

[0148] Paragraph 34. The method of paragraph 22, wherein less than 7,000
cells/cm2 are cultured in step a).

[0149] Paragraph 35. The method of paragraph 22, wherein the specific time
interval is every 108 hours.

[0150] Paragraph 36. The method of paragraph 22, wherein the specific time
interval is every 96 hours.

[0151] Paragraph 37. The method of paragraph 22, wherein the specific time
interval is every 72 hours.

[0152] Paragraph 38. The method of paragraph 22, wherein the specific time
interval is every 48 hours.

[0157] Paragraph 43. The method paragraph 22, wherein the percentage of
tissue stem cells in the population is less than 5%.

[0158] Paragraph 44. The method of paragraph 22, wherein the cell number
at the two passages that are performed without any increase in cell
number has declined to less than 40% of the cell number in step a).

[0159] Paragraph 45. The method of paragraph 22, wherein the cell number
at the two passages that are performed without any increase in cell
number has declined to less than 30% of the cell number in step a).

[0160] Paragraph 46. The method of paragraph 22, wherein the cell number
at the two passages that are performed without any increase in cell
number has declined to less than 20% of the cell number in step a).

[0161] Paragraph 47. The method of paragraph 22, wherein the cell number
at the two passages that are performed without any increase in cell
number has declined to less than 10% of the cell number in step a).

[0162] Paragraph 48. An in vitro method of culturing a heterogeneous
population of cells that allows for determination of the effect of an
agent on the tissue stem cells within the heterogeneous population
comprising: performing sequential passaging of a heterogeneous population
of cells comprising tissue stem cells, transient cells, and terminally
differentiated cells, in a manner that the cells in culture cease to
divide within a period of 100 days (within 90, 80, 60, days), thereby
allowing the number and cell kinetics of tissue stem cells, transient
cells and terminally differentiated cells to be monitored within the
population.

EXAMPLES

Example 1

An Example Passage Schedule for the Invention

[0163] What follows is an example of serial culturing schedule that
embodies the principles of the invention. The starting cell number,
vessel size, dilution amount, and dilution interval may be varied to
achieve efficiencies of time and scale. However, strict adherence to the
time interval fixed dilution schedule such that the cells never reach
more than 50% confluency at the time of any passage in the sequence so
that a decline in cell number is observed (e.g. to less than 50%, 40%,
30%, 20%, or 10% of original cell number) in a period of time until at
least two intervals have occurred without increase in cell number are
important requirements. For example in a period of time less than 100
days, (e.g. less than 90 days, or less than 80 days). The decline in cell
number occurs faster than one would observe in cell cultures of primary
cells that are passaged using a schedule based on cell number, which is
typically corresponds to ≦50 cumulative population doublings
resulting in about 150-200 days of culture before they stop dividing. The
less dense population of cells (i.e. so the peak cell number at the time
of passage results in never greater than 50% confluency) results in a
cell culture that rapidly declines in cell number, as compared to
recommended cell culture passaging schedules for primary cells, because
the cells are not densely populated and for optimal cell growth the cells
like to be close to one another.

[0164] 1. Set-up all 6 wells of a six-well plate with 65,000 viable cells
in each well with 5 mLs total medium in each well.

[0165] 2. Culture for 96 hours.

[0166] 3. At the end of the 96-hour culture interval, each well should be
trypsinized, respectively, and 1/3 of its cells transferred (within a 1
mL volume of culture medium) to a respective well of a new 6-well plate
with 5 mLs of culture medium. Each 6-well culture's passaging is
consistently maintained respectively, distinct of others. (If a
suspension culture is being used, cells are simply removed for counting
and dilution.)

[0167] 4. The remaining 2/3 cells are used for counting as soon as the new
plates are completed and returned to the incubator. Minimize the amount
of time that the transferred cells are in non-ideal conditions.
Thereafter, the cell counts should be conducted immediately to minimize
loss of viability due to non-ideal conditions. Do not put the cells for
counting on ice or chill. Generally, a coefficient of variation
≦5% is required for individual well counts. Both viable and total
counts should be determined.

[0168] 5. The new cultures are grown for 96 hours and the 1/3
dilution-counting procedure repeated.

[0169] 6. This "culture 96 hours, 1/3 dilution, count" procedure should be
continued until two successive passages result in no increase in cell
number.

[0170] The culture schedule described herein is distinct from the
conventional manner in which primary human cell cultures have been
maintained previously. All primary human tissue cultures contain the
three main cell kinetics categories of tissue cells, as illustrated in
FIG. 1, which include rare tissue stem cells, abundant transient
amplifying cells (which also include lineage-committed progenitor cells),
and abundant terminally differentiated cells.

[0171] Computer-simulation can be used to illustrate the significant
differences in the conventional human cell culture schedule and the
invention. FIG. 2 shows a comparison of the progression of total cell
number for cell cultures that only differ for their culture schedule. The
output of the invention schedule is compared to that of the conventional
schedule for human tissue cells. In the conventional schedule for human
cells, investigators wait until cultures reach confluency (˜5
million cells in the simulation for a 75-cm2 flask) before transferring a
fixed fraction of the cells (8). In another schedule used for rodent
cells, a number of cells equivalent to the starting number is transferred
at a fixed interval (9). Therefore, the transfer fraction varies. The
invention schedule differs from both by dictating that a fixed fraction
of the cells is transferred at a fixed interval, no matter how many cells
are present at the end of each growth interval.

[0172] As result of this distinction, conventional cultures continue to
grow for a longer period (>85 days in the simulation) than cultures on
the invention schedule (≦85 days in the simulation), which is
designed to effect a more rapid dilution of tissue stem cells.

[0173] FIG. 3 shows how this difference in culture schedule is reflected
in distinct population doubling outputs, including the continued growth
of the conventional culture (for as many as 150 days). The
computer-simulation in FIG. 4 shows how the difference in culture growth
is related to differences in the rate of tissue stem cell dilution, which
underpins the ability of the invention to distinguish the cell
type-specificity of toxic or activating agents.

Example 2

Determination of the Cell Type Specificity of Agents

[0174] Computer-simulation can be employed to demonstrate that the
invention schedule can be used to determine if any, and which, of the
three different cell kinetics types of tissue cells are killed by a test
agent.

[0175] FIG. 5 shows a computer-simulation of a primary human liver cell
culture estimated to have an initial stem cell fraction of 0.035 (10).
Investigators are able to determine the peak cell number of the serial
culture before each 1/3 dilution, which occurs every 96 hours
(equally-spaced vertical lines). These values are used to calculate the
cumulative population doubling kinetics used for culture comparisons.

[0176] FIG. 6 shows a computer-simulated deconstruction of the total cell
number data into component percentages of tissue stem cells (NS %),
transient cells (NT %), and terminal cells (NT-Terminal %). After an
initial increase in their percentage, tissue stem cells are loss from
culture altogether by systematic cell kinetics dilution. The terminal
cell fraction initially equilibrates to approximately half of the total
cells present, as dictated by the universal tissue cell hierarchy.
However, soon after the loss of all tissue stem cells, all the remaining
cells in the culture progress to terminal cells. This progression is due
to the absence of tissue stem cells that are required for the production
of transient cells, which are the precursors for terminal cells.

[0177] FIG. 7 compares the effects of two different classes of drugs on
serial culture cell kinetics. At its IC50 (concentration that gives 50%
inhibition), a drug that causes the death of transient cells and terminal
cells causes a detectable and characteristic, but modest, change in the
culture cell kinetics (dotted line). In contrast, a stem cell toxic drug,
also at its IC50, induces profound qualitative and quantitative changes
in the cell kinetics output. FIG. 8 provides a computer-simulated
deconstruction to show the specific effect of the stem cell toxic drug to
reduce stem cell number, which accelerates tissue stem cell dilution.

[0178] FIG. 9 provides a comparison of the effect of an agent that has
transient cell-specific toxicity to one that has tissue stem
cell-specific toxicity, both at IC90. The distinction is very clear; and
by comparison to the control culture, the signature of the transient
cell-specific toxin is discernible. FIG. 10 show that the invention
schedule can also delineate agents that are only toxic to tissue stem
cells from ones that are toxic to both tissue stem cells and transient
cells. The essential dependence on tissue stem cell dilution is manifest
by the culture treated with the dual toxic agent exhibiting greater
doubling times, which seems counter-intuitive. However, because the dual
toxic agent reduces transient cell number, it also reduces the rate of
tissue stem cell dilution. This counter effect leads to better overall
culture growth compared to an agent that only kills tissue stem cells,
even though with the same efficiency.

[0179] FIG. 11 shows that the invention schedule can also distinguish
agents that have specific toxicity against terminally differentiated
cells from those with specific toxicity against transient cells. In
addition to a different pattern of deviation from the control signature,
the culture treated with the terminal cell-specific toxin doubles to a
higher value than the control. This counter-intuitive result is another
positive overall growth effect that manifests a reduction in the rate of
tissue stem cell dilution, in this case by eliminating terminal cells.

[0180] In each example, it is the innovation of evaluating cultures in a
decline phase of the culture that allows these important distinctions to
be made.

[0191] Using a pre-existing dataset developed with control human
mini-tissue cultures and cultures treated with the purine nucleoside
xanthosine as the test agent, we have validated the ability to use basic
cell culture data, described in the methods herein, to detect effects on
liver tissue stem cells. It is known in the art that xanthosine increases
self-replication by adult tissue stem cells leading to their exponential
expansion. We used xanthosine as the agent to expand human liver stem
cell strains and to produce human liver mini-tissues. The FIGS. 14A to
14D depicts data of the cultures treated with xanthosine and demonstrates
very clearly xanthosine's effect of inducing self-replication by liver
tissue stem cells. Thus, culture systems of cells comprising
heterogeneous cell population of stem, transient, and differentiated
cells can be used to evaluate the activity of a test agent against tissue
stem cells by using the methods described herein.

[0192] The methods described herein can accurately predict and quantify
the tissue stem cell activity of drug candidates (test agents). The
validation of the methods used to predict growth curves allows for
assessment of the effect of an agent on the cells. The validation was
performed against well-known serial culture data in the cell biology
literature8,9, existing human liver mini-tissue culture data, and
recently developed human lung tissue cell culture data. The ability of
computer software10 to simulate the varied unspecified data sets is
excellent. To obtain serial culture data specified to the inputs designed
for the simulation, serial culture studies were conducted with a
commercially available human lung cell strain that contains tissue stem
cells (WI-38).7,11 The supporting computer software provided an
excellent simulation of the actual data obtained (See FIGS. 13 and 14).
Thus, the methods and comparisons described herein can be used to
accurately determine whether or not an agent is active against stem cells
and/or transient cells and/or terminal cells in human culture. These data
indicate that the technology can be applied to any tissue type having
tissue stem cells, e.g. liver, lung, pancreas, heart, skin, etc. Positive
or negative effects on cell growth can be determined.

[0193] Background

[0194] Because tissue stem cells are responsible for renewing and
repairing human tissues, drugs that interfere with their function or
cause their death are particularly toxic. FIG. 1 illustrates the
universal, hierarchal human tissue cell kinetics architecture. Tissue
stem cells (NS) subtend tissue turnover units comprised of many dividing
and differentiating transient amplifying cells (NT) and terminally
differentiated cells (NT-Terminal). As differentiated terminal cells age,
expire, and are lost from their tissue, they are replaced by the division
of transient cells, which are in turn replaced by the division of the
resident tissue stem cells.

[0195] Despite the clear importance of tissue stem cells in adverse toxic
drug effects, currently there are no pre-clinical assays for general
tissue stem cell toxicity that do not require animals. Even animal
testing is indirect, as it involves evaluating the pathological
consequences of tissue stem cell toxicity (e.g., organ and tissue
failure, cell hypoplasia, tissue dysplasia). Also, animal models are
known to be poor predictors for cell toxicity in humans.12 So, even
laboratory animals are used as a last resort before initiating Phase I
clinical trials for human drug safety evaluations.

[0196] A number of factors conspire to cause the current lack of direct
pre-clinical assays for tissue stem cell toxicity. Because of their
unique place in the universal cell kinetics hierarchy of human tissues,
tissue stem cells are a minute fraction of any human tissue cell
preparation. As a result, they have proven difficult to isolate in
sufficient number or purity to establish reliable assays. For the same
reason, biomarkers for tissue stem cells with sufficient specificity to
quantify tissue stem cells for drug toxicity testing are still not
readily available.13

[0197] The methods described herein circumvent these longstanding barriers
of isolation and identification of tissue stem cells. It does so by
exposing tissue stem cells directly to tested drugs in the context of
fresh or limited-cultured human tissue cell preparations. The
rate-limiting factor for the long-term cell production of any mammalian
cell culture is directly related to the number, viability, and health of
tissue stem cells in the culture. As shown in FIG. 1, since all transient
cells progress ultimately to non-dividing terminal cells, continued cell
production by any human cell culture absolutely depends on the continued
presence of the proliferative function of tissue stem cells in the
culture.

[0198] Because of the asymmetric self-renewal of tissue stem cells, if
tissue cells are serially cultured, cell production eventually stops
because of the inevitable dilution of the tissue stem cell number in the
culture to zero.1-7 Conventional serial culture involves growing a
cell culture until the culture vessel is replete with cells. When
replete, the cells are harvested; and a fixed fraction of the harvested
cells is transferred to a new culture vessel. The new culture is allowed
to grow until replete again, and the dilution process is performed again.
There are well known examples of such serial culture schedules for both
human cells8 and rodent cells.9 In the case of human tissue
cell cultures, this serial process inevitably leads to a complete
stoppage in new cell production. At this endpoint, the cultures contain
only terminal cells. This outcome results first from dilution of tissue
stem cell number to zero, followed by completion of the remaining
transient cells differentiation and production of terminal cells.7

[0199] By altering the serial culture schedule, it is possible to relate
the total cell output of a culture containing tissue stem cells to the
relative number, viability, and quality of tissue stem cells present as
they decline due to dilution. Herein, we employ a serial culture schedule
that does not wait for cell cultures to become replete with cells.
Instead, the specified schedule of culture dilutions is maintained no
matter what cell number is obtained at the end of each culturing
interval. Even when the cell number appears fixed, the dilution is
continued until there are at least two successive culturing intervals
without any increase in cell number. The cell kinetics of such a schedule
can be related directly to the number, viability, and health of tissue
stem cells during the duration of the serial culture. It can also be
related to the cell kinetics activities of transient cells and terminal
cells.

[0200] By comparing the cell kinetics (i.e., cumulative cell population
doublings [CPD] versus time) of control cultures to drug-treated
cultures, it is possible to determine whether an agent is toxic to tissue
stem cells, transient cells, terminal cells, or any and all combinations
of the three cell types. Conversely, it of course follows that the method
can also identify agents that act on tissue stem cells, transient cells,
or terminal cells to increase their division, viability, or function.

[0201] Validation Results

[0202] The PSCK modeling and computer simulation approach, as described
herein in order to detect tissue stem cell toxicity, is based on the
guiding principle that all primary human tissue cell cultures contain the
three main cell kinetics categories of tissue cells, as illustrated in
FIG. 1, which include rare tissue stem cells, abundant transient
amplifying cells (which also include lineage-committed progenitor cells),
and abundant terminally differentiated cells.

[0203] The PSCK simulation describes the progression of these cell
kinetics relationships during serial passaging in culture prescribed by
investigator-entered design variables. These design variables include
cell kinetics factors for all three classes of cells, including their
frequency, viability, and cell division rates, as well is passaging
factors such as splitting basis and splitting interval. We employed
multi-parameter fitting algorithm that allows us to find the set of
design variables that provides the best simulation of experimental data
sets. Once the design variables for the best-fit PSCK simulation are
established, the PSCK software allows us to "deconstruct" specific
quantitative information for each of the three cell kinetics types.

[0205] FIG. 13 shows a comparison of the replicate WI-38 serial passage
data (n=6) to their PSCK simulation. Importantly, the simulation models
very well the characteristic variability of serial passage data. Although
the six cultures were developed by ideal replicate sampling from a single
initiating culture, their serial CPD data exhibit differences in slope
and arrest times (FIG. 13A, arrow).

[0206] However, all show the culture arrest that is characteristic of all
pre-senescent human tissue cultures (FIGS. 13A and 13C, arrows). The PSCK
simulation captures this essential property with a high degree of
confidence (Compare FIG. 13C to FIG. 13A).

[0207] FIG. 13D illustrates the special advantage of the PSCK software
employed for quantitative analysis of the invention data. Each element of
the simulation can be evaluated independently (i.e., "deconstructed").
Deconstruction provides an estimate of live tissue stem cell number at
any time during serial culture. As predicted, the simulation shows that
tissue stem cell number declines rapidly with serial culture, resulting
in the arrest of the serial cultures.

[0208] 2. Validation of the Invention's Ability to Detect the Effects of
Stem Cell-Active Agents

[0209] The ideally specified WI-38 study showed that the method of the
invention could be employed to estimate stem cell number in serial
cultures. The next important validation was to show that the invention
could detect the effects of tissue stem cell-active agents. As an initial
test of this ability, we used existing data sets from serial culture of
our human liver stem cell strains.14,15 Because these analyses were
not ideally developed for the PSCK computer simulation format, they
represented a more challenging validation.

[0210] The validation agent used was xanthosine, a purine nucleoside that
we have shown independently to induce tissue stem cells to switch from
asymmetric self-renewal divisions to symmetric divisions that lead to
self-replication. The induced self-replication, which is reversed when
xanthosine is removed, is the basis for our technology for producing
human mini-tissues.1,15 As shown in FIG. 14, although the
unspecified data are not ideally simulated quantitatively, the PSCK
simulation software captures the essential qualitative differences in the
data sets (Compare FIG. 14B to 14A).

[0211] The control culture exhibits arrest after less than 30 population
doublings, whereas the xanthosine-supplemented culture continues to
exceed greater than 80 population doublings. Deconstruction of the
simulation validates detection of the predicted increase in tissue stem
cell number (FIG. 14C) as a result of increased symmetric
self-replication divisions (FIG. 14D) in response to xanthosine.

[0212] The presented analysis of existing data from earlier xanthosine
studies indicates that the method of the invention can be used to
identify test agents active against tissue stem cells. We used the PSCK
software10 to simulate the predicted effects of toxic drugs on
serial cultures of the human lung cells, which we have independently
shown to contain asymmetrically self-renewing tissue stem cells.7

[0213] FIG. 15 shows four simulated conditions compared to the
experimental mean CPD data developed in FIG. 13. Two idealized cell
type-specific drugs were considered at their IC90. Compared to the
control drug-free condition (FIG. 15A), a transient cell-specific toxic
drug had modest effects on the long-term culture proliferative rate even
at IC90 (FIG. 15B). In contrast, a stem cell-specific toxic drug, at its
IC90, dramatically reduced the extent of culture proliferation (FIG.
15C). This effect was also detected at its IC50 (FIG. 15D).

[0214] Deconstruction analyses showed the specific effects on the two
respective drug types on tissue stem cells (FIG. 16A to 16D). The
transient cell-specific toxic drug at IC90 did not induce significant
changes in live stem cell number (Compare FIG. 16B to 16A). However, both
at IC90 (FIG. 16C) and IC50 (FIG. 16D), the tissue stem cell-specific
toxic drug induced marked reductions in tissue stem cell number. These
simulation analyses indicate that the method of the invention will have
significant power to detect stem cell-specific toxic drug effects.